The fate of phytanic acid when administered to rats

BIOCBIMICAET RIOPHYSIC. ,ICT:\

178

PRELIMINARY

NOTES

The fate of phytanic

acid when administered

to rats

KLENK AND KAHLKE~ reported in 1963 that methylhexadecanoic acid) was present in substantial

phytanic acid (3,7,rr,r5-tetraamounts in the tissues, blood

and urine of humans afflicted with the rare disease referred to as REFSUM’S syndrome2,“. Subsequent investigations by KAHLKE 4+ established the generality of this finding, and supporting

evidence

has been furnished

by RICHTERICH et al.6, and recently

by

STEINBERG et ~1.~ and by HANSEN~. The same acid has also been isolated and identified in trace quantities in certain food substances, namely butterfatg-13, ox fatl”*lj, ox serum 16-la, and sheep fat IQ and in crude petroleum *O. With a view to elucidating the fate of dietary phytanic

acid in mammals

in which litter mates of inbred Wistar specified

amounts

of phytanic

lipids of the livers, kidneys, well as the synthetic

a series of experiments

acid by stomach hearts,

phytanic

brains,

tube. The fatty

“carcases”*,

acid administered)

matography using an ionisation L liquid phases. Identification

acids from the total

serum, faeces and urine (as

were analysed

for 3,7,14,15-tetramethylhexadecanoic

fats9J0J3m15>19, and from post-mortem techniques The

which included phytanic

hydrogenation chromic bulking indicated

of selected

to the rats

to be of purity

yielded phytanic

> 99%.

data

and identified

by

from phytol

by

i3,8.

was prepared

of the resulting

dihydrophytol

by KARRER AND EPPRECHT”~. Fractional

esters, low temperature

fractions,

chromatography

tissues*,

spectrometry

followed by oxidation

acid as recommended

ia vaczLo of the methyl

chro-

acid isolated from ruminant

XEFSUM’S syndrome

mass and infrared

acid administered

with PtO,,

by gas-liquid

detector and polyethyleneglycol adipate and Apiezonof the synthesised phytanic acid, and that extracted

from rat tissues, blood, urine and faeces was based on gas-liquid earlier established

was carried out

rats fed a low-fat diet ad lib., were administered

crystallization

distillation

from acetone

acid which gas-liquid

with

and the

chromatography

It had iodine value 0.0, and its optical

rotatory

dispersion curve showed [CD]-4.1 at 3~2 m,u, -3 at 400 m,u and -2 at 500 rnp. In the first experiment, 0.5 g phytanic acid per day was administered to a group of rats for 4 successive days and the animals were then maintained on the standard low-fat

diet. I rat died after receiving only 3 doses of phytanic acid, 3 died on the 5th day of the experiment, I on the 6th and I on the 8th day. 2 of the survivors of this experimental group -were sacrificed on the 6th day. The rats that died and also those that were sacrificed, contained phytanic acid in amounts ranging from approx. 12% to approx. 76% of the total fatty acids of the kidneys, “carcases”, hearts, and livers, and from approx. 1% to approx. 5% of the total fatty acids in the brains. In the control animals, as in all control animals in the present investigation, this acid was not detected. When 2 of the group of rats that received 0.5 g phytanic acid per day for 4 successive days were maintained on the standard low-fat diet for

* The term “carcase” in this communication denotes the whole rat excluding liver, kidneys, heart and brain. Biochine.

Biophys.

Acta,

116 (1966)

178-18~

PRELIMINARY

NOTES

I79

the following 22 days and then sacrificed, traces only (0.60.9%) of phytanic acid were found in the fatty acids of the livers, hearts and brains, while in the “carcases” and kidneys this acid was absent. In the second experiment, a group of 5 rats was fed 0.1 g phytanic acid per day for IO days and sacrificed on the 11th day. The fatty acids from the kidneys, livers, brains, and hearts contained from 0.3% to 1.8% of phytanic acid, and the “carcases” contained none. Phytanic acid was also present in the fatty acids from samples of urine and faeces from this group, indicating that the animals were eliminating it from their bodies. To a further group of 5 rats 0.1 g phytanic acid per day was administered for IO days and the animals were then maintained on the standard low-fat diet for 22 days before being sacrificed. Examination of the fatty acids of the livers, hearts, “carcases”, kidneys, and brains failed to reveal the presence of phytanic acid in any of these tissues. I rat which was administered 0.5 g phytanic acid per day for 2 days and then maintained on the standard low-fat diet for a further 43 days before being sacrificed, was found to contain a small amount (0.6% of total fatty acids) of phytanic acid in the brain but none in the liver, kidneys, heart or “carcase”. This relatively delayed removal and consequent accumulation of phytanic acid suggests a lower mobilisation or catabolic rate in the brain than in the other organs. In the first experiment reported, the extremely rapid incorporation of approx. 73% of phytanic acid into the fatty acid composition of the livers of the 2 rats that were sacrified was reflected mainly in substantial decreases in the content of palmitic and stearic acids, the figures changing from approx. 34% for each of these 2 constituents in the controls to approx. 2% for palmitic acid and approx. 3% for stearic acid in the experimental animals. The fatty acid composition of the livers of these z experimental animals calculated on a phytanic acid-free basis and compared with that of the controls, showed a marked increase in the content of the unsaturated fatty acids, with oleic acid increasing from approx. 10% to approx. 28%, linoleic acid from approx. 13% to approx. 25% and arachidonic acid from approx. 4% to approx. 17%. The foregoing experiments indicated that when large amounts of phytanic acid were administered to rats, the animals were unable to metabolise or eliminate all of it, with the result that there was an accumulation of phytanic acid in the tissues. Eventually these rats died. When small amounts were administered daily, the rat metabolised or excreted this acid and apparently suffered no adverse affects. STEINBERG et al.zz~z3 demonstrated that the rat rapidly converted phytol to phytanic acid and that phytol (and/or its metabolic products) was readily oxidised to CO,. Similarly, the transformation of phytol to phytanic acid in a REFSUM’S syndrome

patient

labelled

phytol

was established

by STOFFEL AND KAHLKE 24. By orally administering

to a REFSUM’S syndrome

patient

and to a control subject,

STEINBERG

et al.7 showed that more than 80% of the dose was absorbed in both subjects, but

whereas in 12 h the normalsubject oxidised26.~~/~ to 14C0,, the subject with REFSUM’S disease oxidised only 2.7%. The work reported in this present communication is in accord with the findings of STEINBERG et al.zz+, and supports the hypothesis that REFSUM’S disease is associated with an impairment phytanic whether

acid22-24p7. The it was ingested

rapid

oxidation

or injected

in the normal ability to metabolise

of phytol

intravenously

in the rat was found

and suggested

Biochim.

Biophys.

to occur

to STEINBERG~~ that Acta,

116 (1966) 178-180

the conversion was apparently not due to enzyme activity of the intestkl flora. However, it has been established by !VAXABAYASHI AND SHIMAZONO zSthat an enzyme system is present in the mammalian liver which effects oxidation by the introduztion of an hydroxyl group on the omega-methyl carbon, and EXBJAR~-~~ considers that in humans with REFSUM'S syndrome a defect exists in their capacity to oxidise phytanic acid at the omega position. The authors are indebted to Dr. I?. M. SCOPES, Westfield College, University of London, for the optical rotatory dispersion measurements reported in this communication.

E. E(LENK AND W. KAHLKE, 2. Physiol. Chewt., 333 (1963) 133. S. REFSUM, Acta Psychiat. Stand. Sup$l. , 38 (1946) I. S. REFSUM, World Neurology, I (rgrio)334. VV. KAHLKE, K&n. Wochschr., 41 (1963) 783. vv’. KAHLKE, K&n. Wochschv., 42 (1964) 1011. R. RICHTERICH, W.KAHLKE,P.VANMECHELEN ANDE.RossI,KZ&Z.. Wochschr.,4r(r963)800, D. STEINBERG, J. AVIGAN, C. MIZE, L. ELDJARN, K. TRY AND S. Rs~sv~~,Bioclaem.Bio~i~ys. I&s. Conzmupa.,19 (1965) 783. 8 R.P. Ha~s~N,Bioc~~~.Biop~ys. Acta, 106(1965) 304. 9 I% P. I(ANsEN AND F.B. S~O~~N~,~~oc~e~. J.,~o (1951) 358. IO R. I?.HANSEN AND F.B. SYORLBND,B~OG~~~~.J.,~~ (1953) 662. II N. BJURSTAM, B. HALLGRBN, R. REIYAGE AND S. STHLLBERG-ST~~~HACEN, referred to 3y E. STXYHAGEN, 2. Awl. Chem, 181 (1961) 462. 12 W. SONNEVELD,~'. HAVERKAMP BEGENZANN, G. J-VAN BEERE, R. KEUNINGAND J. C.&L SCNOGT, J. Lipid Res., 3 (x962) 351. 13 R.P.HANsEN,F.B. SHORLAXD AND J.D.MoRRIsoN,~.D&'~ lies,, 32 (I96.j)21. rq R.P. HANSEN, F.B. SHORLAND AND K. J. COOKE, J. Sci.FoodA&v.,~ (1958) 391. 15 R. P. HANSEN, Chem. Ind. (London), (rgtig) 303. IG W. R. H. DUNCAN AND G. A. GARTON, Biochem. J., 89 (1963) 414. 17 A. N. LOUGH, Biochem. J., 86 (1963)14P. 18 A.K.LouGH, Biochem.J., 91 (x964)584. xg R. I?.HANSEN, New Zealand J. Sci., 8 (1965) 158. 20 J. CASOX AND D. W. GRAHAM, Tetrahedron, 21 fIg@j) 471. zx P. KARRER AND A. EPPRECHT,N&. Claim. Acta, 23 (1940) 272. 22 D. STEINBERG, J. AVIGAN, C. E. MIZE AND J. H. BAXTER, Fede~a~~on Pvoc., 24 (1965) 290. 23 D. STEINBERG, J. AVIGAN, C. E.&&e AND J. H. BAXTER, Baochena.Bio;bh?is. ~o~.~~~~, 19 (1965) 412. 24 W. STOFFEL AND W. KAHLKE,~~O&TTZ. Biophys. Res.Commzan, rg (1~65) 33. 25 D. STEINBERG, personal communication. 26 K.W~KA~AYASHI ANU'DN. SHIMAZONO, Biochim.Biophys. Acta, 70(1g63) x2. 27 L. ELDJARN, Scand. J. Cl&. Lab. Invest., 17 (1965) 178. I

z 3 4 5 6 7

Received August qth, Biochim. Biophys.

I&,

Acta, 116 (1966)178-180